Fusarium Wilt Resistance Identification and Genetic Analysis of Momordica charantia L. Germplasm

Ziji LIU, Gongfu DU, Yu NIU, Yan YANG

Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences, Danzhou 571737, China

Abstract Fusarium wilt seriously affects the yield and quality of Momordica charantia L. Breeding resistant varieties is the most economical and effective measure to control Fusarium wilt in M. charantia L. This study had comprehensively conducted pathogen identification of Fusarium wilt, resistance evaluation of M. charantia L. germplasm to Fusarium wilt, and genetic analysis of Fusarium wilt resistance in M. charantia L., so as to provide reference for breeding resistant varieties and accelerating the breeding process.

Key words Momordica charantia L., Fusarium oxysporum, Resistance identification, Genetic analysis

1 Introduction

Fusariumwilt caused byFusariumoxysporumis a typical soil-borne disease.F.oxysporumhas a wide range of hosts and a high degree of host specificity. There are 8 specialized types ofFusariumwilt pathogens reported abroad, and there are six specialized types that have been clearly reported in China, includingCucumissativusspecialized type,Benincasahispidaspecialized type,Citrullusvulgarisspecialized type,MomordicacharantiaL. specialized type,Cucumismelospecialized type andLagenariasicerariaspecialized type.

Fusariumwilt inM.charantiaL. is a soil-borne fungus disease caused byFusariumoxysporumSchl. f. sp.MomodicaeSun & Huang. According to reports, the common incidence ofFusariumwilt inM.charantiaL. is 15%-25%, and in severe cases, it is as high as 60%-80%, seriously affecting the yield and quality ofM.charantiaL[1]. In recent years, as the multiple-cropping area continues to expand, the damage ofFusariumwilt inM.charantiaL. is becoming more and more serious. Compared with chemical control measures, breeding and promoting resistant varieties is the most economical and effective measure to controlFusariumwilt inM.charantiaL. This study had comprehensively conducted the isolation and identification of the pathogen ofFusariumwilt inM.charantiaL., the resistance evaluation ofM.charantiaL. toFusariumwilt, and the genetic analysis ofFusariumwilt resistance inM.charantiaL., so as to provide reference for the breeding ofFusariumwilt-resistantM.charantiaL. varieties.

2 Isolation and identification of Fusarium wilt pathogen in M. charantia L.

The isolation and identification of theFusariumwilt pathogen inM.charantiaL. is the basis for resistance identification toFusariumwilt and breeding of resistant varieties. Yang Yeetal.[2]collected 10M.charantiaL. plants infected withFusariumwilt in theM.charantiaL. planting field in Jinhua City, Zhejiang Province, and obtained 23 strains of pathogenic bacteria by tissue separation. According to the morphological characteristics, molecular characteristics, pathogenicity and host specificity of the culture, identification was carried out. The results show that the isolated pathogen wasF.oxysporumSchl. f. sp.MomodicaeSun & Huang[2]. Su Mingxingetal.[3]isolated 19Fusariumstrains fromFusariumwilt-infectedM.charantiaL. plants in Fujian Province, and they were identified asF.oxysporumby Booths method andF.oxysporum-specific primers. After inoculating them into healthyM.charantiaL. seedlings, it was observed that theM.charantiaL. seedlings showed symptom of withering;F.oxysporumwas present in both moderately and severely infected plants; moderately infected plants had the highest bacterial content, followed by severely infected plants; the base of the stems had the highest bacterial content, followed by roots and the middle part of the stems; and the pathogen was not isolated from the top of the stems and the leaves. Chen Zhendongetal.[4]measured the morphological characteristics and host specificity of 32 isolates ofFusariumwilt-infectedM.charantiaL. plants, and the results show that the isolatedFusariumwilt pathogens were allF.oxysporumSchl. f. sp.MomodicaeSun & Huang; these strains could infectM.charantiaL. andLagenarialeucanthaRusby seedlings, but did not infect other cucurbit crops; and there was a certain correlation between the clusters of strains and geographical sources. TakingFusariumwilt-infectedM.charantiaL. plants from sixM.charantiaL. growing areas in Henan Province as test materials, Guo Kangdietal.[5]isolated the pathogen by tissue separation and conducted identification according to colony and spore morphological characteristics, combined with molecular biology analysis and pathogenicity determination. The results show that the 34 strains isolated were allF.oxysporumSchl. f. sp.MomodicaeSun & Huang.

In addition to morphological and molecular biological methods, the classification and identification ofFusariumwilt pathogens must also be conducted combined with the pathogenicity of the pathogen isolates to different host plants. The results of resistance identification are susceptible to human factors, and the identification results are uncertain. Therefore, it is necessary to establish accurate and rapid detection techniques forFusariumwilt pathogens, in order to achieve early diagnosis of cucurbitFusariumwilt and provide a basis for effective prevention and control of cucurbitFusariumwilt, Guo Kangdietal.[6]had screened out a pair of specific primers. The primers and the detection method had good specificity and high sensitivity forF.oxysporumSchl. f. sp.MomodicaeSun & Huang, and can quickly and accurately detectF.oxysporumSchl. f. sp.MomodicaeSun & Huang from soil and plant samples.

Xiao Changhuaetal.[7]found that the germination rate ofF.oxysporumSchl. f. sp.MomodicaeSun & Huang spores was only about 5% in distilled water, 19% in glucose or sucrose solution, and 96%-100% in the decoction ofM.charantiaL.; the optimum temperature, carbon source and nitrogen source for mycelial growth were 28-30℃, soluble starch and peptone, respectively; and the optimum temperature and pH for sporulation were 28℃ and 6.3, respectively. Liu Yanghuaetal.[8]found that theFusariumwilt pathogens that overwintered on the soil surface and the grass in the field still had a role in disease transmission next year; the pathogens in the paddy field lost their pathogenicity within three months; and the pathogens located 5-10 cm below the vegetable land surface lost their pathogenicity after 6-12 months.

3 Resistance evaluation of M. charantia L. germplasm to Fusarium wilt

There have been some reports on theFusariumwilt resistance screening inM.charantiaL. germplasm. Zhu Tianshengetal.[9]had confirmed that inoculating roots with spore suspension was better for resistance identification ofM.charantiaL. toFusariumwilt, the suitable spore concentration was about 106 spores/mL; the disease investigation should be carried out 9-12 d after inoculation; and theM.charantiaL. varieties in Guangdong lacked high-resistance varieties, withFusariumwilt-resistant varieties accounting for about 14% and highly and moderately susceptible varieties accounting for 86%. Guo Tangxunetal.[10]inoculatedM.charantiaL. withFusariumwilt pathogen by root soaking method, soil soaking method and acupuncture method, respectively. They all could cause the incidence ofFusariumwilt in differentM.charantiaL. varieties, but the incidence of the root soaking method was highest, so it is suitable for resistance identification ofM.charantiaL. germplasm toFusariumwilt. The root soaking method was adopted to evaluate the resistance of eight majorM.charantiaL. varieties at the seedling stage. It was found that theM.charantiaL. varieties of Thailand Big Flesh King, Longevity Big Flesh No.3 and Big Flesh New No.3 were moderately resistant toFusariumwilt, and no highly resistant varieties were found. Luo Fangfangetal.[11]collected 11M.charantiaL. varieties from Guangdong Province. After resistance identification by artificial inoculation in the seedling stage, it was found that Green Prince, Jubao No.2, Hainan Big Flesh, Fenglu and Huayan No.2 were resistant toFusariumwilt, Youlu No.3, Hainan Thick Flesh F1 and Nongdeli were moderately resistant toFusariumwilt, and Dading 103, Cuilv No.3 and Bifeng were susceptible toFusariumwilt. Zeng Hualanetal.[12]used the root-breaking method to evaluate the resistance ofM.charantiaL. germplasm in Sichuan toFusariumwilt. The results showed that among the 23 kinds ofM.charantiaL. germplasm tested, there were 15 kinds ofM.charantiaL. germplasm that reach the level of resistance and medium resistance, accounting for 65.22%. Guan Fengetal.[13]isolated and purified three strains ofF.oxysporumSchl. f. sp.MomodicaeSun & Huang with strong pathogenicity from Jiangxi Province, and identified the resistance of 36M.charantiaL. varieties toFusariumwilt using root soaking method. Among the 36M.charantiaL. varieties tested, 6 varieties were highly resistant toFusariumwilt, 11 varieties were resistant toFusariumwilt, 4 varieties were moderately resistant toFusariumwilt, and 9 varieties were highly susceptible toFusariumwilt. Lin Yongshengetal.[14]evaluated the resistance of 61 kinds ofM.charantiaL. germplasm toFusariumwilt by combining root-breaking method and root irrigation method. The results showed that theM.charantiaL. germplasm with resistance and moderate resistance toFusariumwilt accounted for 24.6%, and theM.charantiaL. germplasm with high resistance toFusariumwilt accounted for 3.3%.

Up to now, the screened germplasm was mostly resistant or moderately resistance, and there were very few materials that exhibited high resistance and immunity. There was still a lack of varieties that were highly resistant toFusariumwilt in production. In order to breedFusariumwilt-highly resistantM.charantiaL. varieties, there was still a need to strengthen the collection, identification and resistance evaluation ofM.charantiaL. germplasm, and excavateM.charantiaL. materials highly resistant toFusariumwilt.

4 Genetic analysis of resistance of M. charantia L. to Fusarium wilt

Identifying the genetic laws of resistance toFusariumwilt, breeding and promoting resistant varieties is the most effective measure to controlFusariumwilt inM.charantiaL. Most of the disease resistance is quantitative trait. Different research materials often have different genetic patterns. Disease resistance can be controlled by major gene, minorgenes or the combination of major gene and minorgenes. The resistance of cucurbit crops toFusariumwilt is more complex, showing the characteristics of genetic diversity.

Taking theFusariumwilt-resistantM.charantiaL. material Thai 4-6 and the susceptible material CN19-1 as the parents, hybrid combination was prepared[15]. Based on the six generations of genetic population, the genetic characteristics of resistance to Fusarium wilt were analyzed by using the major gene+polygene mixed genetic model. The optimal model was 2 pairs of additive-dominant-epistatic major gene+additive-dominant polygene (E-1). The additive effect value of the two pairs of major genes was -13.85. The dominant effect values were 25.58 and 34.26, respectively. The heritability of major genes was 86.03%, 80.34% and 94.25% in BCP1, BCP2 and F2, respectively, indicating that the resistance of the combination toFusariumwilt was mainly controlled by the two pairs of major genes. Taking the disease-resistant parent ‘0417’ and the susceptible parent ‘472113’ as materials, Zhao Xiujuanetal.[16]had studied the inheritance of resistance ofM.charantiaL. toFusariumwilt. The resistance ofM.charantiaL. toFusariumwilt was controlled by a single dominant gene with a broad heritability of 90.78%. For breeding disease-resistant hybrids or parental inbred lines, the disease-resistant plants can be selected from the early generation.

In summary, the genetic research on the resistance ofM.charantiaL. toFusariumwilt has been scarce so far. Therefore, further study on the genetic law ofM.charantiaL. resistance toFusariumwilt can provide reference for the breeding ofM.charantiaL. varieties resistant toFusariumwilt.

5 Discussion

Resistance evaluation ofM.charantiaL. varieties in the field is affected by many factors such as pathogenicity ofFusarium, cultivation and management techniques and planting period. In order to reduce the occurrence ofFusariumwilt, it is recommended to rotate varieties of different types or varieties with different resistance genes and to rotate with non-cucurbit crops. At present, there is a lack ofM.charantiaL. varieties or materials that are highly resistant toFusariumwilt. In the future, it is necessary to identify the resistance of local and wild varieties ofM.charantiaL. for screening out varieties or materials with high resistance, and then breeding excellent resistant varieties through various breeding methods for production. In future research, the loci that control the resistance toFusariumwilt should be mapped combining with modern molecular biology techniques, disease-resistant varieties should be bred using molecular marker-assisted selection technique, and the breeding process will be accelerated.